Covalent inhibition of Bruton?s tyrosine kinase (BTK) is a clinically validated mechanism for treating B-cell malignancies as illustrated by the FDA approval of ibrutinib (I) in mantle cell lymphoma (MCL) in 2013 and acalabrutinib (A), a more kinase selective 2nd generation covalent inhibitor approved in 2017 for second line MCL. However, these 2 agents become ineffective in about one-third of MCL patients because of primary resistance and, to a lesser extent, inhibitor-induced resistance. We have created a 3rd generation covalent BTK inhibitor, SRX3262, with more kinase selectivity than A to further decrease side effects. Moreover, SRX3262 was designed to inhibit potently two additional key cancer targets that are synergistic against BTK: PI3 kinase (PI3K) and the bromodomain protein BRD4. We also demonstrate that blocking these two targets it increases activity towards MCL cell lines and overcomes both primary and inhibitor-induced resistance versus I or A. This approach is significant because it will dramatically expand the patient population likely to respond to and benefit from our 3rd generation BTK inhibitors beyond the current CLL and MCL populations. A key innovative component of our proposal is our effective utilization of in silico models to design and synthesize SRX3262, the first-in-class small-molecule chemotype that potently inhibits BTK, PI3K and BRD4 in the same cell (preliminary results). Our demonstrated ?proof of concept? with SRX3262 includes: 1) more kinase selective vs 2nd generation BTK inhibitor acalabrutinib, 2) low nM potency and inhibition of all three cancer targets in cell-based assays, 3) 16-34X more potent than I/A on resistant MCL cell lines, 4) 3X more tumor growth inhibition in vivo vs I in mouse MCL xenograft model, 5) 10X less toxic on normal epithelial cells vs combination of three separate inhibitors (BTKi/BRD4i/PI3Ki). This proposal will further characterize our lead SRX3262 while obtaining a back-up molecule for Phase II development with a focus on MCL. Specific Aims: Aim (Task) #1. Develop a 3rd generation BTK inhibitor with improved efficacy against resistant B-cell malignancies. Approach: Evaluate and characterize SRX3262 for early ADME and tox. Aim (Task) #2. Identify SRX3262 back-up candidate. Approach: In parallel to Aim 1, model in silico a 1,260-membered virtual library of SRX3262 analogs against BTK, synthesize predicted top 15 BTK inhibitors, obtain their BTK inhibition profile, test them against resistant B-cell malignancy cell lines, and obtain key ADME parameters. Aim (Task) #3. Demonstrate superiority to Ibrutinib (I) and Acalabrutinib (A). Approach: Compare toxicity (normal B-cells/epithelial cells) and efficacy of SRX3262 (or back-up) vs I/A in mouse tumor models and cellular assays including cysteine-to-serine resistant cells and intrinsic resistant cell lines. The results of this work will set the stage for a Phase II SBIR focusing on the development of SRX3262 or improved analog through pre-IND studies.